The fermentation of E. coli for the commercial production of recombinant proteins has increased significantly in recent years. Although there have been improvements in the productivity of these processes with regard to protein expression, there remains an opportunity for improvement in the fermentation processes. In particular, the production of acetate as a fermentation byproduct is a common problem. Acetate is undesirable because it retards growth even at concentrations as low as 0.5 g/L, and it inhibits protein formation. Moreover, acetate production represents a diversion of carbon that might otherwise have generated biomass or protein product.
In E. coli, acetate is synthesized mainly by the phosphotransacetylase-acetate kinase pathway (pta-ackA), using acetyl-CoA as the substrate or from pyruvate by PoxB. Acetate is produced under oxygen-limited culture conditions or during aerobic growth with a high concentration of glucose in the medium. These conditions cause an imbalance between the glycolytic and the TCA cycle fluxes, resulting in the excretion of acetate and other metabolites. There have been a number of attempts to apply metabolic engineering to reduce carbon flow to acetate-producing pathways. However, most of the previous approaches have not completely eliminated acetate production, have a deleterious effect on growth rate, or lead to undesirable pyruvate accumulation.
The main sugar uptake system in many bacteria is the phosphoenolpyruvate sugar phosphotransferase system (PEP-PTS), which mediates the uptake and phosphorylation of carbohydrates. The PEP-PTS is a group translocation process where the transfer of the phosphate moiety of PEP to carbohydrates is catalyzed by the general non-sugar-specific proteins enzyme I and HPr in combination with sugar-specific enzyme II (EII) proteins. After autophosphorylation of enzyme I at the expense of PEP, enzyme I catalyzes the phosphorylation of HPr at histidine 15, resulting in HPr (His-P). The phosphate group from HPr (His-P) is then transferred to the sugar substrate via a two-step phosphorylation reaction mediated by a dedicated EII protein. EII proteins can consist of one or more proteins and are composed of three domains: the EIIA and EIIB domains, which are involved in the phosphotransfer, and the membrane-located EIIC domain, which is most likely involved in the translocation of the sugar substrate. The genes encoding HPr and enzyme I, ptsH and ptsI, respectively, have been cloned from several bacteria and often found to be organized in single operon—ptsHI. Several exemplary genes from a variety of microbes are listed here, and more are available in the public databases:
J02796: E. coli ptsH, ptsI and crr genes encoding cytoplasmic proteins of thephosphoenolpyruvate:glycose phosphotransferase system (HPr, enzymes I and Glc-III), completecdsAE008764: Salmonella typhimurium LT2, section 68 of 220 of the complete genomeX12832: Bacillus subtilis ptsX, ptsH and ptsI genes for enzyme III-glucose (EC 2.7.1.69), Hprprotein and enzyme I (EC 2.7.3.9) of PEP:sugar phosphtransferase systemAY750960: Leuconostoc mesenteroides strain SY1 ptsHI operon, complete sequence; and metal-dependent protease-like protein gene, partial cdsZ97203: Lactococcus lactis cremoris ptsH and ptsI genesAY064171: Streptococcus thermophilus pts operon, complete sequenceU12340: Bacillus stearothermophilus XL-65-6 phosphoenolpyruvate-dependentphosphotransferase system glucose-specific permease (ptsG′) gene, partial cds, HPr (ptsH),enzyme I (ptsI), and PtsT (ptsT) genes, complete cds, and wall associated protein precursor(wapA′) gene, complete cdsAF291428: Lactococcus lactis subsp. lactis ptsHI operon, complete sequenceAF316496: Staphylocccus xylosus ptsHI operon, complete sequence; ubiquinol oxidase (cbdA)gene, partial cds; and unknown genesAF172726: Lactobacillus sakei histidyl phosphocarrier protein (ptsI) gene, partial cds; and AtkY(atkY) and copper P-type ATPase AtkB (atkB) genes, complete cdsAF159589: Lactobacillus casei ptsHI operon, complete sequence; and putative sugar permeasegene, complete cdsU82366: Lactobacillus sake ptsHI operon: histidyl phosphocarrier protein HPr (ptsH) and enzyme I(ptsI) genes, complete cdsNC_000913.2: Escherichia coli K12NZ_AAMK01000008.1: Escherichia coli 101-1 Ecol1_01_8NC_007606.1: Shigella dysenteriae Sd197NC_002695.1: Escherichia coli O157:H7 str. SakaiNC_009792.1: Citrobacter koseri ATCC BAA-895NZ_ABEK01000001.1: Salmonella enterica subsp. enterica serovar Agona str. SL483NC_009436.1: Enterobacter sp. 638NC_009648.1: Klebsiella pneumoniae subsp. pneumoniae MGHNC_009832.1: Serratia proteamaculans 568NC_008800.1: Yersinia enterocolitica subsp. enterocolitica 8081NC_007712.1: Sodalis glossinidius str. ‘morsitans’NC_004547.2: Erwinia carotovora subsp. atroseptica SCRI1043NC_008570.1: Aeromonas hydrophila subsp. hydrophila ATCC 7966NZ_AAOJ01000004.1: Vibrio angustum S14 1099604003201
Expression of E. coli ptsHI operon appears to be regulated at the transcriptional level, since mRNA levels were found to be higher in glucose-grown cells than in cells grown on non-PEP-PTS sugars. The PEP-PTS in E. coli generates an excess amount of pyruvate during periods of high glucose uptake exceeding the needs of cell growth, which increases carbon flux towards acetate. Previous investigators have reduced acetate accumulation through the deletion of ptsG, which encodes for EIIglc membrane-bound transporter. Others have completely inactivated the PEP-PTS by deleting the entire ptsHIcrr operon (Lévy, 1990), and utilizing GalP (galactose permease) and Glk (glucokinase) activities for glucose uptake (De Anda 2006).
Although De Anda, Flores and associates were able to inactivate the ptsHIcrr operon, the mutant ΔptsHIcrr bacteria did not grow rapidly and protein production was reduced due to poor bacterial growth and increased acetate production (Flores, 2005a; Flores, 2005b; De Anda, 2006). Acetate production was increased due to the additional GalP and Glk mutations that allowed glucose transport and phosphorylation. Additionally, these bacteria required minimal media to reduce acetate production, further slowing bacterial growth and biomass accumulation.
Therefore, a method of improving ΔptsHI bacterial growth while maintaining low acetate production and PEP required for bio-production is still required.